One of the major problems in understanding antigenic variation in Giardia is the multiple copies of identical or almost identical vsp genes and inability to known which of the family of highly related vsps is being expressed or not expressed. In previous experiments, HA tagged VSPH7 was integrated into the genome of its own host, GS, and underwent antigenic variation identical to native VSPH7. Analysis indicated that integration occurred into a site that differed from vspH7 but nevertheless was virtually identical up to about 3kb. Despite the similar upstream regions, vspH7 and vspH7HA were expressed independently in the absence of gene movement, definitely proving that epigenetic control of antigenic variation in Giardia. During the past year, chromosomal precipitin assays of the immediate upstream region of VSPH7 HA were performed using trophozoites expressing or not expressing the tagged VSP. An anti-acetylated lysine Mab was able to precipitate immediate upstream chromatin from expressing but not from non-expressing trophozoites confirming epigenic control of antigenic variation. Because of technical reasons we were unable to compare other nearby regions but nevertheless the differences between expressing and non-expressing organisms implicates the 5? UTR of VSPs as control region of expression. However, it remains unknown how two identical upstream regions are controlled independently. The downstream. 3? UTR regions of vspH7 HA and vspH7 were sequenced and found to be identical for at least 1 kb downstream confirming the virtual identical immediate surrounding sequence for vspH7 and vspH7HA.Earlier studies from this laboratory examined the regulated expression of cwp 1 and 2. We showed that about 60 bp upstream was required for regulated expression and that the promoter contained inhibitory as well as stimulatory elements. Encystation in Giardia is triggered by cholesterol deprivation that occurs physiologically as Giardia travel down small bowel and at the same time bile and cholesterol are absorbed. In higher eukaryotes the transcription factor SREBP plays an important role is regulating lipid and cholesterol transcription. To determine if similar control mechanisms occur in Giardia, we transfected the upstream promoter region of cwp2 driving luciferase into Chinese hamster ovary cells. Despite the lack of known SREBP binding motifs, absence of cholesterol in the medium or inhibition of cholesterol synthesis by inhibition of HMG CoA activity stimulated the reporter construct while over expression of SREBP or presence of cholesterol in the medium inhibited reporter gene activity. Analysis of the upstream region showed 3 novel SREBP binding motifs. Although these experiments suggest the presence of SREBP or homologous transcription factors in Giardia none could be identified in Giardia by DNA by a number of methods.